508 identification of an alternative specificity for engineered t cells expressing an enhanced affinity MAGE a3 TCR

508 Identification of an Alternative Specificity for Engineered T Cells Expressing an Enhanced Affinity MAGE A3 TCR Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society[.]

CANCER - IMMUNOTHERAPY II 506 CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy Refractory Acute Leukemia Renier Brentjens,1,2 Marco L Davila,1 Isabelle Riviere,2,3 Jae Park,1 Xiuyan Wang,3 Shirley Bartido,3 Jolanta Stefankski,3 Oriana Borquez-Ojeda,3 Yvette Bernal,1 Michel Sadelain.1,2 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY; 2Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY; 3Cell Therapy and Cell Engineering Facility, Memorial Sloan Kettering Cancer Center, New York, NY Adult patients with relapsed B-acute lymphoblastic leukemia (ALL) have a dismal prognosis Only those patients able to achieve a second remission, ideally with no evidence of minimal residual disease (MRD) after salvage chemotherapy, have a meaningful hope for long-term survival after subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT) We have treated a cohort of relapsed B-ALL subjects with autologous T cells expressing a CD19specific CD28/CD3 dual-signaling chimeric antigen receptor (CAR) termed 19-28z All patients with persistent morphological disease or MRD upon T cell infusion demonstrated rapid tumor eradication and achieved MRD-negative complete remissions as assessed by deep sequencing PCR Therapy was well tolerated although significant cytokine elevations, observed in those patients with morphologic evidence of disease at the time of treatment, required additional lymphotoxic steroid therapy These results demonstrate the potency of 19-28z CAR modified T cells and the reliability of this novel therapy to provide a bridge to a potentially curative allo-HSCT 507 A Vaccinia Virus Armed with Interleukin-10 That Dampens Host Immunity to the Vctor but Not to Tumor Is a Promising Therapeutic Agent for Pancreatic Cancer Louisa S Chard,1 Eleni Maniati,1 Pengju Wang,2 Jonathan Hughes,1 Jiwei Wang,2 Bela Denes,3 Istvan Fodor,3 Thorsten Hagemann,1 Nicholas R Lemoine,1 Yaohe Wang.1 Barts Cancer Institute, Queen Mary University of London, London, United Kingdom; 2Sino-British Research Centre for Molecular Oncology, Zhengzhou University, Zhengzhou, China; Center for Health Disparities & Molecular Medicine, Loma Linda University, Loma Linda Vaccinia virus (VV) has several inherent features that make it particularly suitable as an oncolytic virus (OV) for cancer treatment including the most aggressive pancreatic cancer For OV-based therapies the immune system may represent a barrier to efficacy as a vigorous immune response will lead to rapid viral clearance before they are able to effectively eliminate tumor cells To address this issue, we have developed a tumor-specific VV (by deletion of the thymidine kinase (TK) gene) armed with a cytokine interleukin-10 (IL10) IL10 is commonly regarded as a pro-tumor cytokine due to its well-known immunosuppressive effects and involvement in regulatory T cell (Treg) activation However, evidence has accumulated to suggest that IL10 also has immunostimulatory properties and it has been reported to enhance the effectiveness of a Poxvirus-based vaccine in an experimental murine tumor model We therefore hypothesized that the dual properties of IL10 might make it a promising therapeutic candidate gene for cancer therapy Preliminary in vivo experiments comparing the efficacy of VVTK-IL10 to an unarmed virus (VVTK-LacZ) have shown that IL10 significantly improves the anti-tumor efficacy of VV-based treatment, resulting in animals bearing pancreatic cancer becoming 100% tumor-free (compared to 40% of VVTK-LacZ treated animals) 40 days after intra-tumoral injection of the viruses Strikingly, VVLTK-IL10 resulted in a superior anti-tumor activity compared to an unarmed S196 virus (VVLTK) in a K-ras-p53 mutant-transgenic pancreatic cancer model Interestingly, upon rechallenge with the same tumor cells, all the animals previously treated with VVTK-IL10 rapidly rejected the tumor cells, implying the development of tumor-specific immunity within the host Clearance of VVLTK-IL10 was reduced at early time points compared to control virus making it more able to exert oncolytic effects and release more TAAs to the immune system The efficacy of treatment by VV was dependent on CD4+ and CD8+, but not NK cells Treatment with VVLTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8+ cells compared to VVLTK, which accounts for the delay in viral clearance and improved efficacy IL10 reduced the MHCIIhi macrophage populations in tumors, suggesting that intratumoral macrophages were responsible for presentation of viral, but not tumor, antigens These results suggest that VVLTK-IL10 has strong potential as an anti-tumor therapeutic for PaCa and can prevent disease recurrence 508 Identification of an Alternative Specificity for Engineered T Cells Expressing an Enhanced Affinity MAGE A3 TCR Namir J Hassan,1 Brian Cameron,1 Andrew Gerry,2 Joseph Dukes,1 Jane Harper,1 Vivekanandan Kannan,1 Frayne Bianchi,1 Francis Grand,1 Giovanna Bossi,1 Joanna Brewer,2 Debbie Sutton,1 Annelise Vuidepot,1 Alex Powlesland,1 Alison Legg,1 Katherine Adams,2 Alan Bennett,2 Nick Pumphrey,2 Gwen Binder-Scholl,2 Irina Kulikovskaya,3 Minnal Gupta,3 Gabriela Plesa,3 Michael Kalos,3 Bruce L Levine,3 James L Riley,3 Angel Varela-Rohena,3 Edward A Stadtmauer,3 Aaron P Rapoport,4 Gerald P Linette,5 Carl H June,3 Bent Jakobsen.1,2 Immunocore Ltd, Abingdon, Oxfordshire, United Kingdom; Adaptimmune Ltd, Abingdon, Oxfordshire, United Kingdom; Abramson Cancer Center and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 4The Greenebaum Cancer Center, University of Maryland, Baltimore, MD; 5Siteman Cancer Center, Washington University School of Medicine, St Louis, MO Adoptive T cell therapy is emerging as a leading new technology for the targeted destruction of tumor cells in vivo Advances in T cell transduction methodology and TCR engineering, have enabled this approach to become a feasible option for treating cancer We developed an affinity enhanced TCR (a3a) directed to a HLA-A1 restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy MAGE A3 belongs to the family of cancer testis (CT) antigens and is considered an ideal target for adoptive therapy given its re-activation in various tumors and limited expression in normal tissues Pre-clinically, a3a T cells potently killed MAGE-A3 positive cancers and gave no off-target concerns when tested against a variety of normal primary cells However, clinical administration of a3a T cells resulted in fatal toxicity against cardiac tissue (see companion abstract “Cardiovascular toxicity and Titin cross-reactivity of affinity-enhanced TCR-engineered T cells against HLA-A1 restricted MAGE-A3 antigen”) MAGE-A3 or related MAGE proteins were not detected in cardiomyocytes, in normal cardiac tissue or in heart autopsy tissue by quantitative RT-PCR Further in vitro investigations did not reveal a3a cross-reactivity against an expanded array of standard normal cardiac cells However, a3a T cells killed a beating cardiomyocyte culture derived from induced pluripotent stem (iPS) cells Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) encoded by the TTN gene was identified as an alternative target for the HLA-A1 restricted MAGE A3 TCR Titin is elusive in standard tissue cultures but was confirmed to be expressed in iPs cardiomyocytes; the peptide was also identified to be naturally processed and presented by mass spectrometry Thus a3a recognition of Titin was the most likely cause of off target in vivo toxicity These results indicate that recognition of similar but Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy OLIGONUCLEOTIDE & RNAI THERAPEUTICS unrelated peptides presents the most pertinent safety risk for T cellbased therapies and have highlighted additional preclinical screening assays which may help to mitigate this risk in future clinical trials Oligonucleotide & RNAi Therapeutics 509 Targeted, Multifunctional RNA Therapies for Advanced Cancers of the Prostate Luiza I Hernandez,1 William M Rockey,2 Craig A Howell,1 Justin P Dassie,1 Frank J Hernandez,1 Xiu Y Liu,1 Paloma H Giangrande.1,2,3 Internal Medicine, University of Iowa, Iowa City, IA; 2Radiation Oncology, University of Iowa, Iowa City, IA; 3Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA Current therapies for the treatment of castration resistant prostate cancer (CRPC) have shown only limited efficacy The efficacy of these therapies is limited by the molecular complexity of this aggressive disease as well as unwanted side-effects that result from the treatments themselves Thus, new therapies are needed to reduce off-target effects while addressing the need to inhibit multiple molecular targets Presently available non-targeted chemotherapy regimens inhibit multiple targets via drug cocktails Unfortunately, this approach multiplies unwanted side-effects to non-target tissues and reduces the amount of each drug that can be safely administered to the patient To overcome many of the obstacles with current non-targeted drug treatments, we have developed targeted, multifunctional RNA-based reagents directed against aggressive cancers of the prostate This was accomplished by selectively targeting two or more molecular pathways involved in CRPC with dual-function, inhibitory RNAs The first inhibitory RNA (aptamer A9g)¹ targets prostate specific membrane antigen (PSMA), a prostate cancer (PCa) cell-specific surface receptor involved in migration and invasion of prostate cancer cells The second inhibitory RNA (siRNA) is directed against genes known to be involved in growth and survival of CRPC cells (androgen receptor, PI3K/Akt/mTOR, survivin) We demonstrate the efficacy of these reagents in inhibiting PSMA enzymatic activity (linked to carcinogenesis) and subsequent proliferation and migration of CRPC cells in culture Using a novel cell-based assay (RNA-RIP assay), we confirm the ability of these reagents to effectively access the cytoplasm of target cells and deliver their therapeutic cargo to components of the RNAi machinery Furthermore, the CRPC-specific multifunctional RNAs were coupled to near infra-red (NIR) dyes to determine their disposition in vivo (biodistribution/pharmacokinetics) in mouse models of metastatic disease Toward this end, we verify specific targeting of these reagents to prostate tumors expressing PSMA, following intravenous administration Importantly, we demonstrated that, when administered systemically, the CRPCspecific multifunctional reagents reduce metastatic lesions in two independent models of prostate cancer metastasis (bone and lung) In conclusion, these preclinical studies highlight the feasibility of this approach and establish a framework for developing improved reagents for the treatment of human CRPC Furthermore, these studies have yielded general methods for the establishment of a platform therapeutic technology for treating several other human malignancies, which could benefit from targeted, combinatorial gene silencing Rockey, W M et al Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling Nucleic Acid Ther 21, 299-314, doi:10.1089/nat.2011.0313 (2011) Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy 510 Circulating Plasmid Detection in a Phase I Clinical Trial of Intratumoral Injection of pbishRNA™ Nanoplex Christopher M Jay,1 Donald D Rao,1 Zhaohui Wang,1 Neil Senzer,1,2 Phillip B Maples,1 John Nemunaitis.1,2,3,4 Gradalis, Inc., Dallas; 2Mary Crowley Cancer Research Centers, Dallas; 3Medical City HCA, Dallas; 4Texas Oncology, P.A., Dallas STMN1 is a microtubule regulating protein, previously described by our group and others, as a critical cell-division control protein widely over-expressed across a variety of cancers We subsequently demonstrated effective knockdown of STMN1 protein using our proprietary pbi-shRNA™ technology in vitro and in vivo Favorable toxicology and biodistribution studies in biorelevant animals paved the way for a Phase I clinical study of our novel pbi-shRNA™ Nanoplex The Phase I study was designed as a dose escalation (4 patients per cohort), single intratumoral (IT) injection for advanced cancer patients All patients had an accessible surface lesion for IT injection The patients in cohort were injected with 0.7 mg pbishRNA™ Nanoplex Patients were monitored to assess safety Blood samples were collected from a distal site at baseline and time points (30 seconds, and 10 minutes, 1, 6, and 24 hours) post IT injection Whole blood was extracted for total DNA and then analyzed by qPCR for the presence of circulating pbi-shRNA™ STMN1 DNA The clinical samples analysis was performed by SeqWrite (Houston, TX) with GLP protocols under contract and supervision by Gradalis The assay was first optimized by evaluating different commercially available extraction kits (Qiagen PureGene and Qiagen QiaAmp) and two different qPCR hydrolysis probes The dynamic range of the qPCR assay (10^6 – copy of target) was tested with 100 ng and 300 ng background human genomic DNA (simulating the extracted DNA from patient samples) to evaluate for inhibitory effects on the final results The optimized assay was validated using naïve blood samples and the appropriate spike in controls Having completed accrual for cohort 1, no adverse events were observed and patient samples were analyzed Peak circulating plasmid was detected at either the 30 second or minute time points with steep reduction soon thereafter Residual plasmid DNA can still be detected at the 24 hour time point by the assay, albeit at levels significantly lower than peak values.The second cohort of patients is currently being enrolled and this data set will be analyzed when the cohort is completed The protocol for this second cohort has been modified to include a 48 hour time point to quantify the amount of circulating plasmid at a later time point Quantitative circulating plasmid results for cohort and will be reported 511 Smooth Muscle Cell Targeted RNA Aptamers for the Treatment of Vascular Disease William H Thiel,1 Maysam Takapoo,1 Shuxia Jiang,1 Jennifer Streeter,1 Bojana Stanic,1 Xiuying Liu,1 Francis J Miller, Jr,1 Paloma H Giangrande.1,2,3 Department of Internal Medicine, University of Iowa, Iowa City, IA Proliferative and degenerative alterations of the vascular wall contribute to obstruction of blood flow to vital organs and remain a central causative feature of vascular disease and subsequent organ dysfunction Bypass surgery and percutaneous interventions can restore blood flow but subsequent activation of smooth muscle cells (SMC) often results in restenosis or graft failure While recent advances in drug-eluting stents (DES) have significantly reduced the incidence of stent restenosis in patients, DES have also increased the risk of stent thrombosis This complication results from attenuated endothelium healing due to the nonspecific antiproliferative drugs coating these stents Improving the prognosis of patients with cardiovascular disease awaits treatment strategies that S197 ... cocktails Unfortunately, this approach multiplies unwanted side-effects to non-target tissues and reduces the amount of each drug that can be safely administered to the patient To overcome many of the... when the cohort is completed The protocol for this second cohort has been modified to include a 48 hour time point to quantify the amount of circulating plasmid at a later time point Quantitative... patients per cohort), single intratumoral (IT) injection for advanced cancer patients All patients had an accessible surface lesion for IT injection The patients in cohort were injected with